Gene ontology (GO) analysis and characterization of SERBP1-interacting proteins.

A) Selection of enriched GO terms in Biological Processes, Molecular Function, and Cellular Component according to ShinyGO (27). FE = fold enrichment; FDR = false discovery rate. B) Visualization of associated GO enriched terms in three main regulatory branches: translation, ribosome biogenesis and RNA processing. C) Top represented PFAM domains (42) among SERBP1 interactors with counts of 5 or more and significant fold enrichment (FE) and false discovery rate (FDR). The occurrence of each domain within the total human proteome (ALL) is listed as reference. D) Presence of intrinsically disordered proteins (IDPs) (45) among SERBP1 interactors. E) Distribution of identified SERBP1-associated proteins in reference to the presence of RGG boxes (50) and binding to G-quadruplexes (G4s) (46, 47).

SERBP1 and cell division.

A) Structures relevant to mitosis and pertinent SERBP1-associated proteins. B) Western blot showing SERBP1 knockdown in U251 and U343 cells. C) Multinucleated cells in U251 and U343 control and SERBP1 knockdown (KD) cells after treatment with paclitaxel. Data are shown as means of triplicate counts of 1000 cells ± standard deviation and statistical significance was determined by Student’s t-test. D) Aspect of cells exposed to paclitaxel. On the left staining with anti-α-Tubulin; in the middle staining with DAPI showing an increased number of multinucleated cells after siSERBP1 KD. 100X magnification for detailed visualization of a single multinucleated cell. E) Plot shows the results of a cell viability screening (291 drugs) performed in U343 vs. U343 SERBP1 over-expressing (OE) cells. Red dots correspond to drugs whose impact on cell viability was significantly different between U343 and U343 SERBP1 OE. F) Highlights of the screening showing cell cycle/division and DNA damage/replication inhibitors whose impact on cell viability was smaller in U343 SERBP1 OE in comparison to control.

SERBP1 and splicing.

A) Networks showing splicing factors identified as SERBP1 interactors. B) Distribution of splicing events affected by SERBP1 knockdown according to their type. SE, exon skipping; RI, intron retention; MXE, multiple exclusive exons; A5SS, alternative 5’ splice sites; A3SS, alternative 3’ splice site. C) The bar graphs show the percentage of splicing events affected by SERBP1 knockdown with evidence of SERBP1 binding sites close (<100nt) to regulated splice sites. D) Results of SERBP1 and hnRNPU expression correlation in different studies. E) Overlap between splicing events affected by SERBP1 and hnRNPU KDs. F) Sashimi plots showing examples of splicing events affected by both SERBP1 and hnRNPU knockdowns in U251 cells. The red arrows indicate affected exons.

SERBP1 interacts with PARP1 and influences PARylation.

A) Results of IP-western in U251 cells with control and anti-SERBP1 antibodies confirm SERBP1 interaction with PARP1, NNNCL and SYNCRIP. B) Correlation of SERBP1 and PARP1 expression in different studies. C) Similarity of SERBP1 and PARP1 expression profiles during cortex development according to Cortecon (141). D) PARylation sites observed in SERBP1 protein according to (62, 63). E) PARP1 ADP-ribosylates SERBP1 in vitro. Purified recombinant PARP1 (0.1 µM) and SERBP1 (0.6 µM) were combined in a reaction with or without sheared salmon sperm DNA (sssDNA) (100 ng/µL) and NAD+ (100 µM) as indicated. The reaction products were analyzed by SDS-PAGE with silver staining (left) and Western blotting for PAR (right). F) Venn diagram showing the overlap between SERBP1 interactors, PARylated proteins and PAR binders (62–64). G) Increase of PARylation levels in 293T and U251 GBM cells after H2O2 treatment. H) SBP-SERBP1 pulldown in 293T cells shows increased SERBP1 association with PARP1 after H2O2 treatment. SBP-SERBP1-His detected by His antibody; Flag-PARP1 detected by Flag antibody. I) SERBP1 transgenic expression (mGreen-SERBP1) in 293T cells increased the levels of PARylated proteins.

Shared SERBP1 and PARP1 interactors.

A) Overlap between PARP1 (66) and SERBP1 interactomes and data on PAR binding (64) and PARylation (62, 63) of shared interactors. B) Selection of enriched GO terms (biological processes) related to SERBP1-PARP1 shared interactors according to ShinyGO (27). FE = fold enrichment; FDR = false discovery rate. C) Network showing shared SERBP1 and PARP1 interactors implicated in ribosome biogenesis. D) Proposed SERBP1-PARP1 feedback model; SERBP1 function and association with partner proteins is modulated by PARylation while SERBP1 influences PARP activity.

SERBP1-associated proteins are prevalent in membraneless organelles.

A) Different types of membraneless organelles in a cell and examples of SERBP1-associated proteins present in each structure. B) Membraneless organelles identified in the GO enrichment analysis (cellular component) of SERBP1 interactors (72). FE = fold enrichment; FDR = false discovery rate. C) Bar graph showing the distribution of SERBP1-associated proteins in different membraneless organelles. D) Prevalence of G4 binders (46, 47), PAR binders (64), and PARylated proteins (62, 63) among SERBP1-associated proteins present in membraneless organelles.

SERBP1 is present in Tau aggregates.

A) Representative co-immunofluorescence of SERBP1 and Phospho-Tau (Thr231) in AD brain tissues. Merged channel is represented. DAPI was used to stain nuclei. Magnification 20x and white scale bar: 50 µm. Three different Insets selected from merged channels are represented in zoomed images as merged, SERBP1 (green) and Phospho-Tau (red). B) Western blot showing SERBP1 expression in normal and AD brains and presence of oligomers. C) Venn diagram shows overlap between proteins identified in Tau aggregates and SERBP1 associated proteins (top). Selection of enriched GO terms in Biological Processes according to ShinyGO (27) related to proteins present in the overlap (bottom). FE = fold enrichment; FDR = false discovery rate.

SERBP1 association with PARP1 in Alzheimer’s brains.

A) Representative co-immunofluorescence of SERBP1 and PARP1 in AD and age-matched control brain tissues. PARP1 and SERBP1 are represented in gray while merged images represent PARP1 (red), SERBP1 (green). DAPI was used to stain nuclei (blue). Magnification 20x and white scale bar: 100 µm. Each frame has a zoomed inset representing the detailed distribution of each target. B) Pearson Coefficient (PCC) of co-localizing SERBP1 and PARP1 between in cells of age-matched control and AD brains (CTR vs. AD, **** p<0.001 paired t-test). C) Fluorescence intensity profiles of PARP1 (red), SERBP1 (green), and DAPI (blue) in representative cells from age-matched control and AD brains. Distance is represented in pixels and fluorescence intensity as Grey value obtained using ImageJ FIJI software.